The present application is related to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, a parvovirus mutated structural protein which comprises as least one B-cell epitope heterologous to the parvovirus, a mutlimeric structure comprising the protein, a nucleic acid encoding the protein, a virus or cell comprising the protein, a method of preparing the protein, a medicament comprising the protein, nucleic acid or multimeric structure and its use.
Monoclonal antibody therapies have been one of the most successful therapy forms of new drug developments over the last couple of years in therapeutic fields such as oncology, autoimmune and inflammatory diseases. In monoclonal antibody therapies patients are injected with a specific monoclonal antibody that recognizes the antigen involved in the disease. Antibodies recognize their antigen with the variable domain of the antibody which is also referred to as the idiotype of the antibody.
However, monoclonal antibody therapies also have certain drawbacks. It can be observed that, if the concentration of a specific antibody with one particular idiotype is too high, the patient's immune system develops an antibody response against the idiotype of the therapeutic monoclonal antibody and thereby limits its efficacy. This kind of antibody that recognizes an antibody's idiotype is referred to as an anti-idiotype antibody. In addition, antibodies to monoclonal therapeutic antibodies directed against other parts of the monoclonals often limit efficacy of a passive antibody therapy. Therefore, many of the monoclonal antibody drugs need to be used in combination with the traditional immunosuppression regiments, increasing the overall treatment costs. Furthermore, active suppression of the patient's immune system is detrimental especially, if an intact immune system is required to control the stage of disease such as for oncological indications.
As being a passive vaccination against the target antigen the monoclonal antibody has to be injected frequently depending on the half life of the antibody within the serum of the patient. Therefore, such treatments are expensive and inconvenient for the patients.
An alternative for such monoclonal antibody therapies already exists exemplified by a number of clinical developments using anti-idiotype antibodies as drugs. Such anti-idiotypic antibody therapies are based on the fact (see above) that the patient's immune system can induce an antibody response against the idiotype of an antibody. If one uses a monoclonal antibody expressing a functional imitation of a target epitope (paratope or mimotope) as an idiotype, the patient's immune system will generate a polyclonal antibody response wherein a subset of these antibodies is able to cross-react with the target epitope in the patient. Such antibody expressing a paratope is referred to an anti-idiotypic antibody (based on Jerne's network model of idiotypic relationships (Jerne, 1974, Jerne et al., 1982). Thus, selective immunization with an anti-idiotypic antibody can induce a specific immune response directed against the original antigen (Varela and Coutinho, 1991, Jefferis, 1993, Chatterjee et al., 1994).
Therefore, a vaccination with such an anti-idiotypic antibody actively induces a polyclonal antibody response. As a consequence such anti-idiotypic antibody vaccines have a number of advantages over a passive immunization by a standard monoclonal antibody. There is no antibody response towards the anti-idiotypic antibody that limits its efficacy as exactly this immune response is used as the therapeutic principle. Therefore, it is also not necessary to combine the antibody treatment with an immunosuppression regimen. And further, due to the fact that the anti-idiotypic treatment is an active immunization, the drug only has to be injected from time to time to boost the antibody response generated by the patient himself maintaining a continuous titer of specific antibodies. Additionally, anti-idiotype antibodies induce a polyclonal antibody response against the target antigen that hampers the potential mechanism for resistance to the treatment of e.g. in tumor cells.
However, anti-idiotypic antibody therapies face major disadvantages. The titers of the induced polyclonal antibody response obtained by the vaccination with anti-idiotypic antibodies are often not high enough to establish a beneficial treatment. This is due to the lack of a strong antigen as a vaccine, since antibodies per definition are not very immunogenic. Furthermore, it is difficult to generate specific anti-idiotype vaccines because of this lack of immunogenicity and technical difficulties to identify anti-idiotypic antibodies.
A series of publications describes that an antigen placed in the context of an ordered surface of a viral particle—here a papilloma virus particle—can induce a B cell response that even can abrogate B cell tolerance to such antigen by direct crosslinking the respective B-cell receptor. Bovine papilloma virus-like particles (VLPs) conjugated to an Aβ peptide through biotin were used to generate an immune response against the self antigen Aβ (Li et al., 2004). Further, this group used bovine papilloma virus-like particles having the murine chemokine receptor mCCR5 inserted into an immunodominant site of the viral L1 protein to immunize mice leading to sera with high anti-CCR5 antibody titers despite the fact that CCR5 is a selfantigen. Further, a macaque L1-CCR5 fusion protein was used to immunize pig tail macaques. 4 of the 5 test animals produced CCR5 specific antibodies. In a further approach TNF-α was joined to VLPs by way of a biotin-streptavidin interaction (Chackerian et al., 2001). These VLPs were successful in generating an auto-antibody response in mice, whereas these antibodies bound native TNF-α (U.S. Pat. No. 6,719,978).
Therefore, papilloma VLPs have been shown to be a suitable backbone for the presentation of antigens to the immune system in order to generate strong B cell responses, probably because of their dense, ordered and closely packed array of vaccination epitopes. Due do their exceptionally strong B cell induction papilloma VLPs can be especially useful to overcome B cell tolerance to self antigens.
However, it is questionable if epitopes linked by biotin or inserted by an educated guess can possibly induce the generation of auto-antibodies for a wide range of tolerogens, as advantageous epitopes for vaccination may be three-dimensional and inserted epitopes may refold due to the specific environment of the insertion site. This is especially true for small antigens or individual epitopes, where influences of the viral capsid backbone are more relevant than in case of larger insertions.
Therefore, the problem of the instant invention was to find alternative or even superior methods to identify drug candidates useful as vaccines for the treatment of diseases, especially accessible to antibody therapies that avoid one or more of the above mentioned disadvantages (BPV based VLPs with conjugated or manually inserted tolerogen-derived epitopes).
The problem is solved by a screening method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, the method comprising the steps of (a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, (b) providing a binder for an antigen, (c) selecting at least one parvovirus virion specifically binding to the binder, and (d) identifying (i) the parvovirus mutated structural protein or a mutated part thereof, or (ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein of the parvovirus virion selected in step c).
Parvovirures, especially Adeno-associated virus type 2, are well known in the art as viral vectors for gene therapy (Muzyczka, 1992). Further, the AAV2 structural proteins have been genetically modified to change the cellular tropism of AAV2 and thereby direct the virus to cells or tissues that are under normal conditions not infected by the wild-type AAV2. The first successful retargeting of AAV2 was published by Girod A. et al. (Girod et al., 1999), (WO 99/67393). The authors identified insertion sites for AAV that can be modified e.g. by insertion of short peptide sequences without destroying the capability of the structural proteins to assemble into virions. The insertion of a peptide sequence of choice that is displayed on the surface of the virion then leads to an altered cell tropism that has successfully been tested in vivo (White et al., 2004). The technology has been further developed to be used to reduce the antigenicity of AAV to escape from the immune system of patients that have neutralizing antibodies against AAV (Huttner et al., 2003); (WO 01/05990) and to modify the AAV virion's chromatographic properties to enable the efficient manufacture of AAV vectors for gene therapy (WO 01/05991). This work has been confirmed and further insertion sites have been identified (Shi et al., 2001), especially tables 1-5, page 1708 “Identification of optimal sites for heterologous ligand insertion”; (Shi and Bartlett, 2003), US 2002/0192823; (Wu et al., 2000)).
To improve the technology of retargeting AAV to desired cells or tissues, libraries of mutated structural proteins of AAV have been constructed and successfully used for the selection of AAV clones with altered cell tropism (Perabo et al., 2003, Lieber, 2003, Muller et al., 2003, WO 03/054197).
Parvovirus structural proteins have been known in the past to form virus-like particles that can be used for vaccination purposes. A vaccine containing hybrid recombinant parvovirus-like particles of pig parvovirus (PPV) and canine parvovirus (CPV) containing a CD8+ epitope from the lymphocytic choriomenigitis virus (LCMV) nucleoprotein protected mice against lethal infection with LCMV (Casal, 1999). The same was shown for PPV and CPV virus-like particles (VLPs) containing the C3:T epitope from poliovirus (Casal, 1999). Also B19 structural proteins have been applied in epitope delivery for vaccination purposes. VP-2 capsid proteins of human parvovirus B19 VLPs were used to display linear epitopes of human herpes simplex virus type 1 and mouse hepatitis virus A59 (Brown et al., 1994), U.S. Pat. No. 6,719,978).
However, these attempts have been used only for fairly large pathogenic epitopes and not with tolerogens or small antigens or even individual epitopes, where B cell tolerance has to be broken to have a beneficial effect for the patient.
Screening methods using parvovirus libraries have been previously described in WO 03/054197. Disclosed therein are screening methods to identify parvoviruses with an altered cell tropism. The authors further disclose an immunoselection step using antibodies such as patient sera to remove immunogenic parvoviruses from the pool of viruses (negative selection). However, a selection of a parvovirus virions specifically binding to the binder, e.g. for a virion binding to a therapeutic antibody, was not disclosed, being a positive selection.
Medicaments according to the present invention have numerous advantages over the prior art. The immune system of a mammal is specialized to generate strong antibody responses against viral capsid proteins due to the co-evolution of mammals and their immune system on one hand and viruses on the other hand. Strong antibody responses means titers of 1000 to 100.000 measured in a standard ELISA. Virus-like particles are highly immunogenic due to resemblance of a virus and thereby efficient uptake of such particles by antigen-presenting cells. The size of the virion, the density and symmetric order of B-cell epitopes and the optimal distance of about 50 to 100 Å between any two B-cell epitopes plays a major role regarding very strong T-cell independent B-cell responses mediated by direct cross-linking of the respective B-cell receptor breaking even B-cell tolerance against self-antigens or tolerogens (Szomolanyi-Tsuda and Welsh, 1998, Szomolanyi-Tsuda et al., 1998, Szomolanyi-Tsuda et al., 2000, Szomolanyi-Tsuda et al., 2001, Zinkemagel, 2002, Bachmann et al., 1993).
Taken together, such medicaments are capable of inducing a polyclonal immune response against certain B-cell epitopes that leads to an active immune response resulting in long lasting antibody titers. The multimeric structure of the virion contains a large number of densely packed identical epitopes directly cross-linking the respective receptor on B-cells and, thereby, inducing a T-cell independent B-cell response. The particulate structure of the medicament further supports the immune response via efficient uptake by antigen-presenting cells which activate T-cells finally triggering IgG class switch and hypermutation of activated B-cells, leading to the persistent release of high-affinity IgG antibodies and differentiation of B-cells into memory cells. Using the methods of the current invention such medicaments can easily be screened and produced.
The following definitions explain how the defined terms are to be interpreted in the context of the products, methods and uses of the present invention:
A “structural protein” means a protein that is part of the capsid of the virus. For parvoviruses the structural proteins are generally referred to as VP-1, VP-2 and/or VP-3.
A “mutated structural protein” means a structural protein that has at least one mutation in comparison to the respective structural protein of the wild-type virus.
A “parvovirus” means a member of the family Parvoviridae containing several genera divided between 2 subfamilies Parvovirinae (Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus) and Densovirinae (Densovirus, Iteravirus, Brevidensovirus, Pefudensovirus, and Contravirus) (Fields: Virology, fourth edition 2001, Volume 2, chapters 69 and 70, Lippincott Williams Wilkins, Philadelphia. Preferred parvoviruses are members of the genus Parvovirus, such as AAV1, AAV2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV10, AAV11, AAV12, bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV), and goose parvovirus (GPV).
Preferred parvoviruses are adeno-associated virus (AAV), Bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), minute virus of mice (MVM), B19, H1, AAAV, feline panleukopenia virus (FPV) and goose parvovirus (GPV). Especially preferred are AAV1, AAV2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV10, AAV11 or AAV12, especially AAV2.
The term “binder” refers to a molecule that specifically binds to its respective binding partner. Commonly used binders are antibodies, especially monoclonal antibodies, antibody derivatives such as single chain antibodies or antibody fragments. In principle all classes of antibodies can be used, preferred are IgG antibodies. Fragments or multimers of antibodies can equally be used. Commonly used fragments are single chain antibodies, Fab- or (Fab)2-fragments. Examples of other suitable binders are protein scaffolds such as anticalins or lipocalins (Nygren and Skerra, 2004), receptors or parts thereof (e.g. soluble T-cell receptors), ankyrine, microbodies or aptamers. The term “specifically binds” means that two molecules A and B, preferably a proteins, bind to each other thereby generating complex AB with an affinity (KD=koff/kon) of at least KD=1×10−5 mol/l, preferably 1×10−7 mol/l, more preferably 1×108 mol/l, especially 1×10−9 mol/l.
The term “antigen” in the context of the products, methods and uses of the present invention refers to any target antigen against which an immune reaction should be induced. Such target antigens are usually antigens that are susceptible to the humoral immune response. They are usually proteins that may be posttranslationally modified, as for example glycosylated proteins. Preferred antigens are serum proteins, proteins that can be found at least under certain conditions (e.g. in a disease state) in the blood (e.g. CETP, IL-6, IL-17, TNF-α), and membrane proteins, especially receptor proteins (e.g. CD20, acetylcholine receptors, IL13R, EGFR). Especially preferred antigens are IgE, tumor-antigens (e.g. Melan A, high molecular weight melanoma associated antigen (HMW MAA), CA125, IL13R, Her2/NEU, L1 cell adhesion molecule), VEGF, EGFR, CD20, IL-9, IL-13, CETP (cholesterol ester transfer protein), TNF-family members (e.g. TNF-α), interleukins (IL-6, IL-17) or misfolded proteins leading to a protein aggregation and, therefore, causing conformational diseases (for an overview see Uversky et al., 2006), e.g. β-amyloid). Excluded from the above definition of “antigen” are parvovirus antigens, i.e. antigens inherent the unmutated parvovirus itself, e.g. derived from B19 (Klenerman et al., 2002).
“Heterologous” in the context of the present invention means a peptide sequence, e.g. an epitope that is not present on the parvovirus wild-type viral capsid and/or structural protein.
A “tolerogen” is a self-antigen that is—in its natural environment—accessible to the humoral immune system. It may be either secreted or otherwise released from a living cell or associated to the outer surface of or integrated into the cellular membrane. Generally speaking tolerogens do—under normal circumstances in contrast to e.g. autoimmune diseases—not evoke a specific immune response due to tolerance against the antigen which results from a previous exposure to the same antigen. Tolerance can occur due to central tolerance or peripheral tolerance. Central tolerance refers to tolerogens which corresponding antigens have been exposed to T cells in the thymus leading to elimination of the specific T cells. Peripheral tolerance occurs when antigens/epitopes/mimotopes/paratopes are presented to T cells without appropriate additional stimuli, commonly provided by inflammation leading to anergy. Still, it has been observed that tolerogens can induce to some extent regulatory B-cell responses (Vogel et al., 2004).
In one preferred embodiment this invention relates to tolerogens due to peripheral tolerance, preferably tolerogens derived from tumor antigens/epitopes/mimotopes/paratopes. Tolerogens encompassed by this invention include peptides, nucleic acids, carbohydrates, and lipids, preferably peptides.
Preferred tolerogens are antigens on the surface of a cell, especially tumor cells, e.g. receptors, especially growth factor receptors, tumor antigens, viral receptors, CD20, acetylcholine receptors, interleukin receptors. Further preferred tolerogens can be blood proteins such as interleukins, IgE, cytokines, immunoglobulins, complement factors, CETP and VEGF.
A “tolerogen-derived epitope” of a specific tolerogen in the context of the products, methods and uses of the present invention refers to a B-cell epitope that
i) is identical to a B-cell epitope of the tolerogen,
ii) a derivative (e.g. a mutant) of a B-cell epitope of the tolerogen that crossreacts with an antibody that binds the B-cell epitope of the tolerogen.
iii) a mimotope of a B-cell epitope of the tolerogen, and/or
iv) a paratope of a B-cell epitope of a tolerogen.
The length of a tolerogen-derived epitope is typically 4-30, preferably 5-20 and most preferably 5-15 amino acids.
The derivative of a B-cell epitope of a tolerogen may be generated by one or more amino acid substitutions, preferably one or more conservative amino acid substitutions, i.e. substitutions that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc. Further, derivatives may be obtained by one or more single amino acid deletion(s) and/or insertion(s).
“Crossreaction” or “crossreact” of B-cell epitopes with a specific monoclonal antibody means according to this invention that the affinity (Ko) of the epitopes with the antibody are within two magnitudes, preferably within one magnitude when comparing the B-cell epitope to its derivative.
Tolerogen-derived epitopes within the multimeric structure comprising parvovirus mutated structural proteins according to this invention are identical, resemble or mimic antigen stretches of a tolerogen that are—in their natural environment—accessible to the immune system, e.g. epitopes of membrane protein located in the extracellular part, serum proteins, immunoglobulins, plaque proteins. Such antigen stretches are preferably located on the surface of such protein within the body of a mammal, preferably a human.
A “mimotope” is a non-linear structural epitope composed of several amino acids derived from different regions of the linear sequence of the structural protein located in close neighborhood due to the overall tertiary structure of the capsid that is specifically bound by an antibody, or a linear epitope mimicking a discontiuous epitope of the structural protein.
A “paratope” is the antigen binding site that is specifically bound by an antibody.
The mimotope or paratope in the context of the present invention might consist of (parts of) the inserted peptide sequence alone or might be composed of inserted peptide and parvovirus core particle amino acid residues.
An “insertion” of (an) amino acid(s) is generally speaking an insertion of at least one heterologous amino acid into the sequence of—for this invention—a parvovirus structural protein. ‘Heterologous’ in this context means heterologous as compared to the virus, from which the parvovirus structural protein is derived from. The inserted amino acids can simply be inserted between two given amino acids of the parvovirus structural protein. An insertion of amino acids can also go along with a deletion of given amino acids of the parvovirus sturctural protein at the site of insertion, leading to a complete substitution (e.g. 10 given amino acids are substituted by 10 or more inserted amino acids) or partial substitution (e.g. 10 given amino acids are substituted by 8 inserted amino acids) of amino acids of the parvovirus structural protein.
The invention relates to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, the method comprising the steps of (a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein, (b) providing a binder for an antigen, (c) selecting at least one parvovirus virion specifically binding to the binder, and (d) identifying (i) the parvovirus mutated structural protein or a mutated part thereof, or (ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein, of the parvovirus virion selected in step (c). The identified gene or mutated part thereof can then be expressed in a cell to obtain the parvovirus mutated structural protein or mutated part thereof.
For identification the at least one gene or the mutated part thereof encoding the parvovirus mutated structural protein may be transferred into a cell, and a cell producing the parvovirus mutated structural protein capable of binding to the binder can be identified. The gene or the mutated part thereof encoding the parvovirus mutated structural protein can be cloned by transducing the gene into a cell and a cellular clone producing the parvovirus mutated structural protein capable of binding to the binder may be identified. Additionally or alternatively, the gene encoding the parvovirus mutated structural protein may be sequenced comprising the individual steps of obtaining bound virions, optionally amplifying the DNA contained within the virions, and sequencing. Sequencing can be performed by standard methods, e.g. after PCR-amplification of at least the part of the parvoviral structural protein that contains the insert. Amplification products can be cloned into a plasmid, and the plasmids can be transformed into bacteria. Single clones can be sequenced and this sequence information can then be used to generate AAV particles of clonal origin.
In case of AAV the identified capsid sequences can be cloned into a standard AAV helper plasmid or in a plasmid containing the full AAV genome. For example, the 587 insertion site of AAV2 is flanked by NotI/AscI restriction sites which can be used for subcloning of the identified peptide-coding sequences into different VP expression vectors. Alternatively, a large part of VP3 can be subcloned by BsiWI and a second restriction enzyme cutting the vector backbone 3′ of the cap ORF (e.g. XmaI in pUC19).
In a preferred embodiment the at least one parvovirus virion selected in step c) of the method of the invention is amplified by viral replication and subsequent packaging in a production cell under suitable conditions, wherein at least steps b) to c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, preferably 1, 2, 3, 4 or 5 times. Optionally, a step for coupling genotype and phenotype of a selected mixture of parvovirus virions can be performed as it is described below for the coupling of a whole library.
The general principle of the identification of such a parvoviral mutated structural protein is that selections are done using a library of viruses displaying a random peptide on the capsid surface. Virus capsid mutants which are able to bind the used binder, e.g. antibody, are selected, can be amplified and are re-used for a new selection round. After each selection round the selected sequences can be analyzed. A selected parvoviral mutated structural protein clone and/or its respective nucleic acid sequence is then used to generate a medicament, i.e. a vaccine.
Preferred binders for the carrying out the identification step of the present invention are Fab or (Fab)2 fragments. If whole IgG antibodies are used, the coupling of the antibody to a support such as a culture plate takes place at random so that the library will be exposed to different parts of the IgG antibody (e.g. the desired idiotype, or the large Fc part which is an undesired event). Therefore, AAV particles which do not only bind the antibody idiotype but which also can bind other parts of the IgG antibodies will be finally isolated. This can be avoided by using only Fab or (Fab)2 fragments. If those fragments are commercially not available for a specific monoclonal antibody, they can be generated from whole IgG antibodies by commercial kits.
There are also alternatives how binding can be performed. In one embodiment the selecting step is performed using a binder that is immobilized on a carrier, preferably directly or via a linker. Such linker can again be linked through a second site of the binder (the first site is the site that is used for screening the library) to a support or surface, e.g. of 12-well plate, e.g. using antibodies as binders through an Fc-specific linker such as protein A or G, sepharose. Fc-specific antibodies or -fragments. The binding of the binder through a linker to the surface has the advantage that the binder is bound in a directed fashion that can avoid unspecific binding of parvovirus mutated structural proteins. By this means, mainly the idiotype of the antibody will be exposed to the particles of the library.
Supports or surfaces used for the method of the invention can either be the surface of Petri dish, 12-well plate or alike, but also suitable chromatography material such CNBr-activated Sepharose. In the latter case a batch method using the chromatography material in suspension can be used.
Alternatively, the selection step is performed using a binder in suspension. Here, binder and parvovirus mutated structural proteins are capable of forming complexes in suspension which reflects the situation in vivo best. Such complexes can be precipitated using standard immune precipitation with Fc-specific antibodies or anti antibody affinity chromatography. Further, binders can be captured by Sepharose A/G columns. Instead of standard immune precipitation magnetic beads coupled to the selection antibody or to any binder which binds to the modified parvoviral particle can be used to isolate the desired modifications out of the library pool.
Specificity of the selection step in either way can be enhanced by addition of at least one washing step to remove unbound or weakly bound parvovirus mutated structural proteins. Suitable washing conditions are e.g. high salt concentrations or use of detergents such as Tween.
Additionally, selected parvovirus virion is further selected for non-binding to a second binder. Such binder might for example be derived from antibodies neutralizing the respective core particle. Another class of binder would be antibodies induced by the same parvovirus virion without inserted peptide sequences. By this means of selection, undesired immunodominant epitopes might be excluded.
Transduction of the gene into a cell is preferably carried out under conditions, where the uptake of the DNA is independent of an infection pathway but occurs through unspecific uptake (e.g. pinocytosis or phagocytosis) further described in more detail below.
Alternatively or additionally, the selection step can be carried out on cells expressing a specific receptor for a binder of choice which is used for selecting the desired parvoviral variant. E.g. cells can be used which express the FcγRI receptor which is specific for any binder comprising an Fc-part of an IgG antibody. For this example, such FcγRI expressing cells can be transduced with a library pool of parvoviruses. First, a negative selection can be performed to avoid unspecific selection of parvoviral candidates which by themselves are able to transduce cells independently from an interaction of a binder with FcγRI. Therefore. FcγRI-expressing cells are incubated with the library pool. The supernatant (pool of parvoviruses which is not able to transduce the cells) is collected and subsequently incubated with the binder of choice (e.g. selection antibody) to perform the positive selection. In the positive selection parvoviruses decorated with the binder will be able to transduce FcγRI expressing cells through attachment of the binder to FcγRI on the surface of the cells. The transduced cells can subsequently be used to amplify the particles.
The specific affinity or avidity of the selected parvovirus mutated structural proteins for the binder can be further enhanced by the additional steps of (e) randomizing the gene encoding the parvovirus mutated structural protein, (f) packaging the randomized genes into a further library of parvoviruses and (g) repeating the steps (a) to (d) of the above selection method.
In a preferred embodiment the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild type structural protein e.g. with its mutated amino acid residue either directly or indirectly contributing to the overall avidity or affinity of the respective virus particle to the binder of an antigen due to the formation of structural epitopes, mimotopes or paratopes.
Further mutation of the capsid protein might be adequate to e.g. i) introduce additional or even identical B-cell epitopes of the same target antigen, and/or ii) B-cell epitopes of one or more further target protein(s) (multi-target vaccine), T-helper 1 (TH1) epitope(s) to further promote the desired TH1 immune response, peptide sequence(s) to target antigen-presenting cells, or to obtain capsid mutants with reduced immunogenicity of the core particle. The latter might be one possibility to setup an efficient prime/boost regimen.
In a further preferred embodiment the further mutation might be adequate to introduce at least one cytotoxic T-cell epitope (CTL epitope). For both infectious diseases and cancer it is most useful to combine both humoral and cellular immune responses to fight these diseases. The multimeric structures according to the invention are in principle capable of pseudo-infecting cells. Accordingly these structures—like viruses—are able to enter cells, are processed to peptides, the peptides are loaded onto MHC class I and II molecules and finally presented to CD8- or CD4-positive T cells. The T-cells become stimulated after specific recognition of such processed peptide presented by MHC class I or II molecules. As a consequence of such stimulation CD8 cells may differentiate into cytotoxic T cells and then cause a cellular immune response. CD4 cells may develop into T helper cells which stimulate B cells to provide a humoral immune response or CD8-positive T cells to provide a cytotoxic immune response, which may themselves induce lysis of infected cells and other cells carrying and presenting the same peptide. Suitable CTL epitopes are known in the art for various cancer antigens or viral antigens, or they can be predicted from given antigen sequences using for example the peptide prediction program by Parker. Proposed CTL epitopes can be validated according to the methods as exemplified for HPV-epitopes in U.S. Pat. No. 6,838,084, examples 2-8 (herein incorporated by reference). As processing of CTL epitopes occurs within the cell it is not necessary that such CTL epitopes are located on the surface or are present in a specific conformation.
It is a preferred embodiment of the present invention that an identical B-cell epitope is inserted at two insertion sites, especially in I-587 and I-453, if it is key to have a large number of identical peptides being optimally presented on the surface of a capsid, especially in the case if direct B-cell receptor (BCR) crosslinking is required for T-cell independent priming of B-cells and breaking of tolerance against self-antigens. A higher density of B-cell epitopes increases the likelihood of optimal peptide-specific BCR crosslinking which requires a defined distance between BCRs (e.g. about 5-10 nm), and therefore, respective B-cell epitopes being presented on a parvovirus capsid. As shown in this invention (FIG. 19), identical insertions of—in this case a β-amyloid epitope—into parvovirus capsids at two (or more) different sites at a time can lead to a higher affinity of the capsid to an antibody specifically recognizing the inserted epitope, here the β-amyloid epitope at insertion sites I-453 and I-587. Consequently, in this case it is preferred that the inserted peptide is a B-cell epitope, more preferred a tolerogen-derived epitope. Therefore, it is an especially preferred embodiment of this invention that an identical peptide is inserted at I-453 and I-587 and that this peptide is a B-cell epitope, most preferred a tolerogen-derived epitope.
Further preferred double insertion variants are all possible combinations of I-261, I-453, I-534, I-570, I-573 and I-587, preferably I-261 in combination with I-587 and I-261 in combination with I-453.
Moreover, a larger number of inserted B-cell epitopes decreases the probability for undesired immune reactions against the parvovirus backbone due to i) masking of natural parvovirus B-cell epi/mimotopes and/or ii) slight structural capsid changes rendering these natural B-cell epi/mimotopes less immunogenic. Accordingly, parvovirus structural proteins comprising at least three insertions are especially preferred.
In a preferred embodiment genotype and phenotype of each virion particle of the library is coupled. This means that the genomic mutant of the virion is identical to the phenotypic mutant of the same virion or, in other words, that each structurally modified virus codes for its structural protein mutant.
In contrast to a bacterial transformation, where only one bacteriophage is taken up by one bacterial cell, using transfection methods for eukaryotic cells many DNA copies (up to 1×106) can be taken up per cell (Dean et al., 2005). Therefore, in the case of an AAV library one cell can replicate thousands of AAV genomes at the same time where each may express a different mutated structural protein with a different peptide sequence inserted into VP-1, VP-2, and/or VP-3 of AAV. At least some of these structural proteins can assemble a complete viral capsid (consisting of 5 VP-1, 5 VP-2 and 50 VP-3 proteins) encapsidating only one of the thousands of AAV genomes present in the cell. In case of a geno-/phenotypically coupled library at least 10%, preferably more than 25%, especially more than 50% of the resulting AAV particles have an encapsulated genome which codes for at least 25%, preferably more than 50%, especially more than 80% of the 60 VP proteins of which its capsid is composed. As a consequence, if an uncoupled library was used for a first screening against a target antibody, the chance that screened particles contained the genome coding for this specific peptide sequence might be very low.
In general, geno-/phenotypically coupled virion particles/libraries are obtained when introducing one single copy of the virus genome into each virion production cell entering the cell nucleus. This cell will only produce capsid protein variants encoded of exactly the introduced genome which is replicated and afterwards packaged into the mutant virion particle. Different experimental settings can ensure this:
To obtain a geno-/phenotypically coupled library of parvovirus virions a library of parvovirus virions is produced by transfecting a plasmid library into production cells under suitable conditions whereas a low copy number of viral genomes equal to or less than 100 genomes per cell is used, preferably equal to or less than 10 genomes, more preferably equal to or less than one genome per cell, resulting in geno-/phenotypically coupled virions/library. The overall transfection efficacy will be finally decisive for the ideal number of virus genomes per cell to be transfected.
The required amount of virus plasmid can be quantified, if e.g. autonomous replicating plasmids with similar size as the virus genome encoding a reporter gene such as GFP are used as a model system. Autonomous replicating plasmids are e.g. systems comprising SV40 origin of replication and large T antigen or the EBV (ebstein barr virus) P1 origin and EBNA. Increasing amounts of the self-replicating reporter gene plasmid are cotransfected with carrier DNA such as empty plasmid DNA (e.g. pUC derivates) keeping the amount of total DNA constant. In theory, each cell transfected with the reporter gene plasmid will, due to its self-replication, express sufficient amounts of reporter protein to be detected. At some ratio of reporter gene vector to carrier DNA, a further increase of reporter gene plasmid will lead to a corresponding increase in the number of transfected cells. By this means, the ideal amount of self-replicating reporter gene plasmid can be determined, reflecting the ideal amount of vector genomes.
Similarly, another read-out system for detection of successfully transfected cells are methods such as in-situ PCR to detect the transfected plasmid genome on a single cell level.
Alternatively, the geno-/phenotypically coupled library of parvovirus virions can be produced by transducing a (non- or partially coupled) virion library into production cells under suitable conditions at a ratio of genomes per cell of 5 to 5,000, preferably 10 to 1,000, more preferably 50 to 300, especially approximately 100, and selecting transduction conditions to be independent from infection pathways, particularly through unspecific uptake through pinocytosis and/or phagocytosis, resulting in geno-/phenotypically coupled virions/library. As it is known that a peptide insertion into the I-587 site of AAV2 frequently destroys (dependent of the sequence of the inserted peptide) the heparin binding motif required for efficient infection of HSPG-receptor containing cells such as HeLa or 293 cells, simple infection methods could bias the screening method and lead only to mutants that still can enter HeLa cells specifically through the respective receptor, in case of AAV2 through heparan sulfate proteoglycan (HSPG). Therefore, an unspecific uptake of the virus particle by the production cell is advantageous. Such unspecific uptake can be achieved by seeding production cells on immobilized parvovirus virions. A preferred embodiment relates therefore to a method, whereas the transduction of the parvovirus virion library is performed using production cells seeded on immobilized parvovirus virions. For this method, the virions are directly coated to a support/surface, e.g. a tissue culture plate. Alternatively, first a capsid specific antibody (in case of AAV2 for example A20) is coated to the support/surface and second the capsids are bound to the coated antibodies. The advantage in the latter case is that the antibody/virus particle complex, respectively the virus particle itself is more efficiently detached from the support/surface and thereby internalized by the cell. Importantly, introduction of foreign peptide sequences into I-587 of AAV2 does not destroy the affinity of A20 to the respective mutant particle as the epitopes of A20 are hardly, if at all, affected by the peptide insertion. The cells, e.g. HeLa cells, are finally seeded on the bound capsids. It is expected that this procedure leads to an uptake of the virus, e.g. AAV, by the cell independent of the natural infectious pathway, presumably by pinocytosis and/or phagocytosis.
In a further preferred embodiment a geno-/phenotypically coupled library of parvovirus virions can be obtained by a method where selected virions are specifically taken up by production cells. In this case the library of parvovirus virions is produced by transducing the library into production cells under suitable conditions at a ratio of genomes per cell of 10 to 10,000, preferably 50 to 5,000, more preferably 100 to 3,000, especially approximately 1,000, wherein transduction conditions are selected to be dependent on infection pathways, particularly through specific receptor binding, resulting in geno-/phenotypically coupled virions/library. In order to achieve such receptor-specific uptake the virions of the library are preferably not immobilized but added to the cells in suspension, whereas both cells and virions can be in suspension or cells are immobilized and virions are added in suspension. Therefore, the transfection of the cells is basically dependent on the virus's infection pathway. In this context it is conceivable to transduce FcγRI expressing cells as described above but incubating the selected pool with A20 antibody-decorated AAV particles (instead of incubating the pool with a binder).
Dependence on infection pathways means that virions are taken up by the cells e.g. through receptor-specific uptake, e.g. for AAV2 heparin sulfate proteoglycan (HSPG)-specific uptake (e.g. for virion libraries where natural infection pathways are not blocked or destroyed by the inserted random peptide sequences). For AAV2 particles with peptide sequences being inserted into I-587, infection of cells will work as long as the capsid contains sufficient HSPG binding motifs or binding motifs for secondary receptors expressed on the cell line used for the coupling step. An AAV capsid consists of 60 capsid proteins each containing the I-587 insertion site. Therefore, mosaic capsid virions containing a given percentage of wild-type sequence capsid proteins will still be able to infect cells via HSPG or secondary receptors. Alternatively, virions with peptide insertions partially restoring the affinity to HSPG or secondary receptors will be able to infect cells such as HeLa or 293. Especially peptide sequences containing basic amino acid residues such as lysine or arginine at the correct position have been shown to restore the natural HSPG infection pathway of I-587 AAV capsid mutants. Given the frequency of basic amino acid residues in a 7mer random sequence and given the fact that an AAV capsid consists of 60 capsid proteins, many if not most of the virions of a non-coupled I-587 AAV2 library consisting of particles with a mosaic capsid will still infect cells to a certain degree via HSPG receptor-mediated uptake.
To keep biodiversity of the library during the coupling step (either by transfection of virus genomes or by cell transduction with virion particles by either means, uptake or infection), always an at least 10-fold, preferably 100-fold, especially 500-fold excess of genomic particles compared to the multiplicity of parvoviral mutants should be transduced in order to ensure that each virus variant is amplified. To further ensure that each virus is coupled in the resulting library an at least 2-fold, preferably at least 5-fold excess of cells is to be used compared to total number of genomic particles.
Geno-/phenotype coupling is desired as the genetic information of the packed DNA can easily be used to obtain the sequence of those particles having high affinity or avidity to the respective antigen binder. It is an object of the invention to use for the identification of a parvovirus mutated structural protein such geno-/phenotypically coupled libraries with a coupling of at least 5%, preferably of at least 25% and more preferably of at least 50%, especially at least 90%.
In a preferred embodiment the library has a multiplicity of parvoviral mutants of greater than 105, preferably greater than 106, especially greater than 107. Multiplicity means according to this invention the number of different virions or viral genomes within the library. In principal it is advantageous to use a library of high multiplicity as the likelihood to identify a suitable or even ideal clone increases with the multiplicity of the library.
The multiplicity of the library is generated by insertion of a nucleic acid insert into the coding region of the gene encoding a parvoviral structural protein leading to an amino acids insertion at a particular position within the parvoviral structural protein. It is preferred according to this invention that the insertion(s) is inserted into one or more positions selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, 1-588, I-591, I-657, I-664, I-713 and I-716, more preferably I-261, I-453, I-534, I-570, I-573 and I-587, especially I-587.
The used nomenclature I-### refers to the insertion site with ### naming the amino acid number relative to the VP1 protein of AAV2, however meaning that the insertion may be located directly N- or C-terminal, preferably directly C-terminal of one amino acid in the sequence of 5 amino acids N- or C-terminal of the given amino acid, preferably 3, more preferably 2, especially 1 amino acid(s) N- or C-terminal of the given amino acid. For parvoviruses other than AAV2 the corresponding insertion sites can be identified by performing an amino acid alignment or by comparison of the capsid structures, if available. Such alignment has been performed for the parvoviruses AAV1, AAV-6, AAV2, AAV-3b, AAV-7, AAV-8, AAV10, AAV-4, AAV11, b-AAV, AAV-5, GPV, B19, MVM, FPV and CPV (see FIG. 1).
For example the insertion site I-587 corresponds to an insertion before and/or after one of the following amino acids indicated by emphasis
SEQ ID NO: 1: FQSSS TDPAT of AAV1, SEQ ID NO: 2: LQRGN587 RQAAT of AAV2, SEQ ID NO: 3: LQSSN TAPTT of AAV-3b, SEQ ID NO: 4: LQSSS TDPAT of AAV-6, SEQ ID NO: 5: LQAAN TAAQT of AAV-7, SEQ ID NO: 6: LQQQN TAPQI of AAV-8, SEQ ID NO: 7: LQQAN TGPIV of AAV10, SEQ ID NO: 8: NQNAT TAPIT of AAV11and SEQ ID NO: 9: NQSST TAPAT of AAV-5.
Further, the insertion site I-453 corresponds to an insertion directly N- or C-terminal of the following ten amino acids each, preferably directly C-terminal of the amino acid indicated by emphasis
SEQ ID NO: 10: QNQSG SAQNK of AAV1, SEQ ID NO: 11: NTPSG453 TTTQS of AAV2, SEQ ID NO: 12: GTTSG TTNQS of AAV-3b, SEQ ID NO: 13: QNQSG SAQNK of AAV-6, SEQ ID NO: 14: SNPGG TAGNR of AAV-7, SEQ ID NO: 15: QTTGG TANTQ of AAV-8, SEQ ID NO: 16: QSTGG TQGTQ of AAV10, SEQ ID NO: 17: SGETL NQGNA of AAV11and SEQ ID NO: 18: FVSTN NTGGV of AAV-5.
Relating to the AAV2 sequence insertion sites for AAV and other parvoviruses encompassed by this invention are listed in Table 1.
TABLE 1Insertion sites for parvovirusesInsertioncorresp. aminositeacid/sequence of AAV2ReferencesI-1M1M1 AADGYSEQ ID NO: 19(Wu et al., 2000) I-34P34PPPKP34 AERHKSEQ ID NO: 20(Wu et al., 2000) I-138T138EPVKT138 APGKKSEQ ID NO: 21(Wu et al., 2000,Warrington et al., 2004,Lux et al., 2005) I-139A139PVKTA139 PGKKRSEQ ID NO: 22(Shi et al., 2001, Shi andBartlett, 2003, Arnold et al.,2006) I-161K161SGTGK161 AGQQPSEQ ID NO: 23(Shi et al., 2001, Arnold etal., 2006) I-261S261YKQIS261 SQSGASEQ ID NO: 24(Girod et al., 1999) I-266A266SQSGA266 SNDNHSEQ ID NO: 25(Wu et al., 2000) I-381N381YLILN381 NGSQASEQ ID NO: 26(Girod et al., 1999) I-453G453NTPSG453 TTTQSSEQ ID NO: 11data of this invention I-447R447YYLSR447 TNTPSSEQ ID NO: 27(Girod et al., 1999, Wu etal., 2000) I-448T448YLSRT448 NTPSGSEQ ID NO: 28(Grifman et al., 2001) I-459R459TTQSR459 LQFSQSEQ ID NO: 29(Shi et al., 2001, Arnold etal., 2006) I-471R471ASDIR471 DQSRNSEQ ID NO: 30(Asokan and Samulski,2006, Moskalenko et al.,2000) I-534F534EEKFF534 PQSGVSEQ ID NO: 31(Girod et al., 1999) I-570P570RTTNP570 VATEQSEQ ID NO: 202data of this invention forΔ566-575 I-573T573NPVAT573 EQYGSSEQ ID NO: 32(Girod et al., 1999) I-584Q584STNLQ584 RGNRQSEQ ID NO: 33(Shi et al., 2001, Shi andBartlett, 2003) I-587N587LQRGN587 RQAATSEQ ID NO: 2(Girod et al., 1999, Shi etal., 2001, Maheshri et al.,2006, Ried et al., 2002,Grifman et al., 2001,Nicklin et al., 2001, Arnoldet al., 2006) I-588R588QRGNR588 QAATASEQ ID NO: 34(Shi and Bartlett, 2003) I-591A591NRQAA591 TADVNSEQ ID NO: 35(Wu et al., 2000) I-657P657VPANP657 STTFSSEQ ID NO: 36 I-664A664TFSAA664 KFASFSEQ ID NO: 37(Wu et al., 2000) I-713T713NVDFT713 VDTNGSEQ ID NO: 38 I-716T716FTVDT716 NGVYSSEQ ID NO: 39(Maheshri et al., 2006)
Amino acid 138 is the N-terminus of VP-2. Preferred embodiments are VP-2 structural proteins with an N-terminal fusion to one of the amino acids within the stretch T138 APGKKR (SEQ ID NO: 40) of AAV2 or the corresponding amino acids of other parvoviruses.
I-570 is especially suitable as an insertion site that goes along with a deletion of given amino acids of the parvovirus structural protein at the site of insertion, leading to a complete substitution. In this case the amino acids RTTNPVATEQ can be substituted by an epi- or mimotope.
Further, the inserted nucleic acid sequence may be inserted at any site corresponding to the first amino-terminal amino acids 1 to 50 of VP-1.
Insertions have been successfully made into AAV-serotypes other than AAV2.
TABLE 2Insertions into AAV-serotypes other than AAV2AAVIns. site/amino acidserotypeSequencerelative to AAV2ReferencesAAV1FQSSS588 TDPATSEQ ID NO: 1I-587N587own data AAV1SSSTD590 PATGDSEQ ID NO: 41I-589Q589(Arnold et al.,2006, Stachlerand Bartlett,2006) AAV-3NNLQS586-SNTAPSEQ ID NO: 42I-585R585(Arnold et al.,2006) AAV-4GGDQS584-NSNLPSEQ ID NO: 43I-585(Arnold et al.,2006) AAV-5TNNQS575-STTAPSEQ ID NO: 44I-585(Arnold et al.,2006)
The most preferred insertion sites are:
i) I-587 as various insertions have been made in the amino acid stretch around N587 (LQRGN587 RQAAT, SEQ ID NO: 2) of AAV2. Within this stretch insertions of various peptides were made C-terminal of amino acids Q584, N587, R588 and A591 in AAV2 (Table 1) and C-terminal of amino acids of other AAV-serotypes corresponding to R585 and Q589 of AAV2 (Table 2).ii) I-453 as according to this invention epitopes have been successfully inserted C-terminal of G453 in AAV2.iii) FQSSS588 TDPAT (SEQ ID NO: 1) or SSSTD590 PATGD (SEQ ID NO: 41) of AAV1.iv) I-261 as according to this invention epitopes have been successfully inserted C-terminal of S261 in AAV2.v) I-534 as according to this invention epitopes have been successfully inserted C-terminal of F34 in AAV2.vi) I-570 as according to this invention epitopes have been successfully inserted C-terminal of P570 in AAV2.vii) I-573 as according to this invention epitopes have been successfully inserted C-terminal of T573 in AAV2.
Corresponding amino acids for all insertion sites specified herein for parvoviruses disclosed herein can be retrieved from the alignment in FIG. 1, for those parvoviruses not listed herein an alignment under standard parameters as used herein can be formed with the provided amino acid sequence of such parvovirus and the corresponding amino acids can be retrieved from such alignment.
The amino acid numbers are given relative to the VP-1 amino acid sequence. However, insertions into the structural gene encoding the structural protein may generally also lead to mutated VP-2 and optionally VP-3 proteins comprising an insertion at a site which is corresponding to the VP-1 insertion as VP-2 and VP-3 are generally expressed from the identical structural gene using downstream located start codons for the start of translation leading to—compared to VP-1-N-terminally truncated structural proteins. A schematic organization of the cap gene of AAV2 is provided in FIG. 2. Therefore, the present inventions encompasses structural genes of parvoviruses with corresponding insertions in the VP-1, VP-2 and/or VP-3 proteins. For example for AAV2, insertions into the cap gene between the codons coding for amino acids 1 and 138 lead to an insertion only in VP-1. Insertions between codons coding for amino acids 138 and 203 lead to an insertion in VP-1 and VP-2, whereas insertions after the codon coding for amino acid 203 lead to insertions in VP-1, VP-2 and VP-3.
Preferred insertion sites are the positions following the amino acids that correspond to the AAV2 amino acids number 139, 161, 261, 381, 447, 453, 459, 534, 570, 573, 584, 587, 588, 657 and 713, especially 261, 453, 534, 570, 573, 587, and 588, most preferably 453 and 587. The amino acid numbers are given relative to the VP-1 amino acid sequence of AAV2.
One further embodiment of the present invention are structural proteins of parvoviruses containing insertions within the previously not described insertion sites I-453 and/or I-570.
Using I-453-based libraries may result in the selection of other peptides (as with I-587-based libraries) since adjacent residues may have an influence on the exposure and functionality of the peptides inserted into the structural protein. In addition, the sites (I-587 and I-453) are located on different loops of the AAV capsid. Thus a different mechanism of cell interaction can be assumed. Furthermore, AAV particles derived from I-453 libraries can be purified with heparin affinity chromatography, as the heparin binding site overlapping with I-587 is still intact. The same applies to other insertion sites not overlapping with I-587, preferably insertion sites I-261, I-534, I-570 and I-573.
In one potential embodiment insertions that have been selected in separate screening rounds can be combined with other insertions selected independently. For example one can use a library with an insertion of random peptides at the I-587 site for the screening method and, independently, use a second library with an insertion of random peptides at another site. Selected structural proteins of the two screening methods can then be combined by standard cloning techniques to make one clone that contains the screened insert at the respective two insertion sites.
In a further embodiment, preferred libraries contain multiple insertions at multiple sites of the structural proteins. Especially preferred libraries/structural proteins have insertions in I-453 and I-587.
By designing the sequence of the nucleic acid insert the multiplicity of the library can be controlled. The generation of such a library is for example described in WO 03/054197, hereby incorporated by reference.
The nucleic acid insert has a number of characteristics. It does not, by insertion into the coding region of the parvoviral gene, create a frame shift and thereby a truncated parvoviral structural protein. Therefore, by insertion a multimer of 3 nucleotides is inserted into the coding region of the parvoviral structural gene. The sequence is a randomly or partially randomly generated sequence, thereby generating the multiplicity of the library. A partially random sequence can for example be used to reduce the number of potential stop codons generated by insertion of the sequence and thereby reducing the number of non-functional structural mutant proteins and/or to achieve a more homogeneous distribution of the twenty different amino acids. e.g. by choosing a NNK design (with each N being any nucleotide and K standing for G or T) which in parallel reduces the number of stop codons from three to one.
In a preferred embodiment, the nucleic acid insert may contain, in addition to the randomly or partially randomly generated sequences, a further stretch of at least one codon upstream and/or downstream of the randomized or partially randomized nucleic acid sequences, preferably of 2 to 12 codons coding for small amino acids, preferably Ala, Gly, Ser, Pro, and Cys, especially an insertion of three codons for Ala upstream and two codons for Ala downstream of the randomized or partially randomized nucleic acid sequences, or an insertion of 2-5 glycin residues both, up- and downstream of the randomized or partially randomized nucleic acid sequences. Such additional amino acids do not enlarge the multiplicity of the insertion but may act as spacers to contribute to the proper accessibility of the inserted amino acids at the surface of the virions.
In a further preferred embodiment the insertion comprises linker sequences which enable a circularization of the inserted peptide sequences in order to better present the insertion. Accordingly spacer sequences are selected to form Zinc-fingers (Zn-finger), well known in the art. Preferred Zn-finger motifs are C2H2, C4, and C2HC including but not limited to motifs CX2CXnC2, CX2CX10-30CX2C, CX5HX10-30CX2C, CX2CX10-30CX4H (Laity et al., 2001 and Gamsjaeger et al., 2006).
An example of a preferred Zn-finger linker is:
X(3-5)CXXCX(0-5)(NNK)n X(0-5)CXXCX(3-5)
(X=Gly or Ala, C=Cys; with each N being any nucleotide and K standing for G or T). Thus the random NNK sequence protrudes from the capsid surface.
As B-cell epitopes are composed of at least 4 amino acids (US 2004/0228798A1), in a preferred embodiment the parvovirus mutant structural protein comprises at least one insertion of 4 to 30 amino acids, preferably 5 to 20 amino acids, especially 5 to 15 amino acids. The B-cell epi-, para- or mimotopes might be composed of the inserted sequence alone, or of amino acid residues of both, the inserted peptide sequence and the viral core protein.
In a further preferred embodiment the insertion comprises within the fixed stretches upstream and downstream of the randomly or partially randomly generated sequences at least one cysteine on each side capable of forming a disulfide bond. Such a disulfide bond would spontaneously form and thereby would stabilize a loop consisting of the inserted amino acids between the two cysteines. Such loop facilitates the optimal exposure of the inserted sequence to the antibodies.
It is also an embodiment of the present invention that the parvovirus mutated structural protein comprises at least one further mutation at a different position. Such further mutation can be used to compose more complex mimotopes, to modify certain properties of the virion, e.g. it can be use to modify its natural antigenicity (e.g. (Huttner et al., 2003); WO 01/05990), to modify its chromatographic properties (e.g. WO 01/05991), to insert a second B-cell epitope, preferably a tolerogen-derived epitope, or to insert a CTL epitope. Such further mutation is selected from a point mutation, an internal or terminal deletion, an insertion and a substitution. Preferably, the further (second) insertion is internally or a N- or C-terminal fusion, whereas the further insertion has a length of 4 to 40, preferably of 5 to 30, most preferably of 7 to 20 amino acids. In one specific embodiment the insertion is a tag useful for binding to a ligand. Such tags are well known in the art, examples for such are listed in Table 3.
TABLE 3Tags and corresponding ligandsTagLigandHISNickelGSTGlutathioneProtein AIgGBiotin or StrepStreptavidinCalmodulin-binding peptideCalmodulinFc-Peptide of IgGProtein AFlagGLAG- or 3xFLAG peptideHA (hemagglutinin)HA peptide
In a further preferred embodiment affinity of a identified parvovirus mutated structural protein for the binder can be modified, preferably enhanced, by generating a library of nucleic acids encoding such parvovirus mutated structural protein having a small number of random mutations per nucleic acid, at other sites than the insertion and or within the insertion, and starting the method of identifying a parvovirus mediated structural protein over again. Such process may be repeated several times, preferably 1 to 5 times, especially 1 to 2 times. A small number of random mutations in this context means an average of at least 10 sequenced clones with 1 to 10, preferably 3 to 8, especially 4 to 6 mutations compared to the starting sequence of the identified parvovirus mutated structural protein. Such random mutations can be inserted by standard techniques known in the art such as error prone PCR and DNA shuffling. In order to achieve that, the viral genomes of the mutants will be isolated and cloned into a suitable plasmid backbone. Random mutations are then inserted by e.g. error prone PCR and/or DNA shuffling. After this, a new packaging is done, followed by a genotype/phenotype coupling step and new selection for binding to a binder of choice, e.g. antibody binding.
Another embodiment of the invention is a parvovirus mutated structural protein obtainable by the methods disclosed above.
A further subject of the present invention relates to a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus and not identical to a pathogen, particularly to a B-cell epitope of a pathogen, and wherein the B-cell epitope is located on the surface of the virus.
A preferred embodiment of the invention is a parvovirus mutated structural protein of the invention may be defined as described above in the context of the method of the invention. As used herein the term B-cell epitope is meant to include also mimotopes. Therefore, the epitopes can be both linear or structural. However, especially linear epitopes that are no mimotopes are preferred.
Typically, the size of a B-cell epitope is at least 4 amino acids (US 2004/0228798A1). Therefore, it is a preferred embodiment that the parvovirus mutated structural protein has an insertion consisting of at least one single or multimeric B-cell epitope of 4 to 30 amino acids, preferably 5 to 20 amino acids, especially 5 to 15 amino acids, and a further stretch of at least one amino acid upstream and/or downstream of the B-cell epitope, preferably of 2-12 amino acids selected from the group consisting of Ala, Gly, Ser, Pro, and Cys, especially 3 Ala upstream and 2 downstream of the B-cell epitope, 5 Ala upstream and 5 downstream of the B-cell epitope, or 5 Gly upstream and 5 Gly downstream of the B-cell epitope. It is preferred that such B-cell epitope is not identical to a pathogen, particularly to a B-cell epitope of a pathogen, that—in its natural environment—is accessible to a humoral immune response. Pathogen, according to this invention, means a virus, bacterium and/or eukaryotic parasite.
Such excluded B-cell epitopes of a pathogen can be identified by searching protein databases known to the skilled artisan. If the searched sequence is identical to a sequence present in a protein of a pathogen, such B-cell epitope is, according to this preferred embodiment of the invention, excluded from the invention.
In a further embodiment, the B-cell epitope heterologous to parvovirus is not identical to a mammalian (including human) or pathogen B-cell epitope, but is a functional derivative of a mammalian or pathogen B-cell epitope. A functional derivative is defined as a B-cell epitope that is identifiable e.g. by the methods according to this invention or that crossreacts with a specific monoclonal antibody for such mammalian or pathogen B-cell epitope.
In further embodiments parvovirus mutated structural proteins of the invention are further characterized as defined above, particularly wherein the tolerogen is as defined above.
In an especially preferred embodiment the parvovirus mutated structural protein a comprises a B-cell epitope that is a tolerogen-derived epitope.
Preferably the B-cell epitope is a part of an antigen as defined above. Preferred antigens are IgE, tumor-antigens (e.g. Melan A, high molecular weight melanoma associated antigen (HMW MAA), CA125, IL13R, Her2/NEU, L1 cell adhesion molecule), viral receptors (CCR5), VEGF, EGFR, CD20, IL-6, IL-9, IL-13, IL-17, CETP, TNF-family members (e.g. TNF-α), or β-amyloid.
In a preferred embodiment the B-cell epitope is not a sequence previously inserted into AAV2 at position I-587/I-587 selected from the group consisting of
SEQ ID NO: 45: QAGTFALRGDNPQG.
SEQ ID NO: 46: SIGYPLP,
SEQ ID NO: 47: NGR,
SEQ ID NO: 48: CDCRGDCFC,
SEQ ID NO: 49: RGDAVGV,
SEQ ID NO: 50: RGDTPTS,
SEQ ID NO: 51: GENQARS,
SEQ ID NO: 52: RSNAVVP,
SEQ ID NO: 53: NSSRDLG,
SEQ ID NO: 54: NDVRAVS,
SEQ ID NO: 55: EYHHYNK,
SEQ ID NO: 56: MTPFPTSNEANLGGGS,
SEQ ID NO: 57: QPEHSST,
SEQ ID NO: 58: VNTANST,
SEQ ID NO: 59: NDVRSAN,
SEQ ID NO: 60: NDVRAVS,
SEQ ID NO: 61: VTAGRAP,
SEQ ID NO: 62: APVTRPA,
SEQ ID NO: 63: DLSNLTR and
SEQ ID NO: 64: GQHPRPG, as listed in Table 4.
TABLE 4Insertions at 587/588 of AAV2, which showed enhanced transduction ontarget cells (inserted in I-587 or I-588).sequence around 587/588 of wt AAV2QRGN---------------------RQAAenhancedSEQ ID NO: 65targettransductionRefQRGN-QAGTFALRGDNPQG------RQAAβ1 and β3 integrinB16F10, RN22(Girod et al.,SEQ ID NO: 451999) QRGN-ASIGYPLPA-----------RQAAPeptide selected byHUVEC,(Nicklin et al.,SEQ ID NO: 66phage display onHSVEC2001)HUVEC QRGN-NGR-----------------RQAACD13RD, KS1767(Grifman et al.,SEQ ID NO: 472001) QRGN-ATGCDCRGDCFC---------QAAαvβ3 and αvβ5HeLa, K562,(Shi andSEQ ID NO: 67Raji, SKOV-3,Bartlett, 2003)local appl. invivo QRGN-AAARGDAVGVAA--------RQAAnot knownMO7e(Perabo et al.,SEQ ID NO: 68selected by AAV2003)display QRGN-AAARGDTPTSAA--------RQAAnot knownMO7e(Perabo et al.,SEQ ID NO: 69selected by AAV2003)display QRGN-AAAGENQARSAA--------RQAAnot knownMec1,(Perabo et al.,SEQ ID NO: 70selected by AAVprim. B-CLL2003)display QRGN-AAARSNAVVPAA--------RQAAnot knownMec1(Perabo et al.,SEQ ID NO: 71selected by AAV2003)display QRGQR-GNSSRDLGA-----------QAAnot knownprim. human(Muller et al.,SEQ ID NO: 72selected by AAVcoronary2003)displayendothelialcells;heart aftersystemic appl. QRGQR-GNDVRAVSA-----------QAAnot knownprim. human(Muller et al.,SEQ ID NO: 73selected by AAVcoronary2003)displayendothelialcells QRGN-ASEYHHYNKA----------RQAAnot known, selectedprim. human(Work et al.,SEQ ID NO: 74by phage displaysaphenous2004)on primary humanvein andsaphenous veinarterial SMCSMC QRGN-ASMTPFPTSNEANLGGGSA-RQAAnot known, selectedHUVEC,(White et al.,SEQ ID NO: 75by phage displayvenous endo-2004)on HUVECthelial cellsafter systemicappl. QRGN-ASQPEHSSTA----------RQAAnot known, selectedbrain endo-(Work et al.,SEQ ID NO: 76by in vivo phagethelium after2006)displaysystemic appl. QRGN-ASVNTANSTA----------RQAAnot known, selectedlung endo-(Work et al.,SEQ ID NO: 77by in vivo phagethelium after2006)displaysystemic appl. QRGQR-GNDVRSANA-----------QAAnot knownHSaVEC(Waterkamp etSEQ ID NO: 78selected by AAVal., 2006)display QRGQR-GNDVRAVSA-----------QAAnot knownHSaVEC(Waterkamp etSEQ ID NO: 79selected by AAVal., 2006)display QRGQR-GVTAGRAPA-----------QAAnot knownCalu6(Waterkamp etSEQ ID NO: 80selected by AAVal., 2006)display QRGQR-GAPVTRPAA-----------QAAnot knownCalu6(Waterkamp etSEQ ID NO: 81selected by AAVal., 2006)display QRGQR-GDLSNLTRA-----------QAAnot knownPC3(Waterkamp etSEQ ID NO: 82selected by AAVal., 2006)display QRGQR-GGQHPRPGA-----------QAAnot knownH9C2(Waterkamp etSEQ ID NO: 83selected by AAVal., 2006)displaybold: amino acid sequence of peptide insertion used to target the new receptor;italic and underlined: amino acid used as linker sequence to flank the targeting peptide;bold and double underlined: amino acid has been substituted in comparison to wild-type sequence;B16F10 = mouse melanoma cell line,RN22 = rat schwannoma,HUVEC = human umbilical vein endothelial cells,HSVEC = human saphenous vein endothelial cells,RD = rhabdomyosarcoma,KS1767 = Kaposi sarcoma,HeLa = human cercix carcinoma,K562 = human chronic myeloid leukemia in blast crisis,Raji = Burkitt lymphoma cell line,SKOV-3 = ovarian cancer,MO7e = megakaryocytic cell line,Mec1 = derived from B-cell chronic lymphocytic leukemia in prolymphoid transformation,HSaVEC = primary human venous endothelial cells,Calu6 = lung carcinoma cell line,PC3 = prostate carcinoma cell line,H9C2 = rat cardiomyoblasts
In a preferred embodiment the B-cell epitope is not a selected from the group consisting of an integrin, especially a β1, β3, avβ3 or avβ5 integrin, and CD13.
In a preferred embodiment the epitope is not fused to the N-terminus of the structural protein of the virus, especially not fused to the N-terminus of VP1 or VP2, whereas fusion to the N-terminus of VP3 is envisaged within this invention.
In a preferred embodiment the parvovirus mutated structural protein is capable of inducing an immunoglobulin capable of binding to the antigen the B-cell epitope is derived from.
It is an important feature, that the B-cell epitope is located on the surface of the virus.
In a preferred embodiment of the instant invention the structural protein of a parvovirus as defined above comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/mimotope.
Vaccines for the Treatment of Asthma and Allergic Diseases
Atopic asthma and allergic rhinitis are caused by adverse immune responses, typified by IgE, against otherwise harmless environmental proteins, allergens. In sensitized individuals, allergen-specific IgE becomes localized in tissues by binding to the high-affinity receptor for IgE, FεRI, expressed by mast cells in various tissues and basophils as well as eosinophils in the blood. Subsequent encounters with the allergen result in cross-linking of IgE/FcRI, which triggers effector cell degranulation and the release of both preformed mediators (histamine, proteolytic enzymes, and proteoglycans) and de novo synthesized mediators (prostaglandin D2, leukotrienes, and cytokines). Together, these mediators are responsible for the clinical manifestations of allergic reactions, including hay fever, asthma, and eczema, as well as life-threatening anaphylactic reactions. Standard therapy includes inhaled corticostreroids (ICS), Beclomethasone Dipropionate (BDP), long-acting β-agonists (LABA) and leukotriene receptor antagonists (LTRAs).
The receptor-binding region of human IgE was previously mapped to the N-terminal region of the CH3 domain (Helm et al., 1988. Helm et al., 1989). Site-directed mutagenesis studies to identify the amino acid residues directly involved in the interaction have been conducted on both IgE (Presta et al., 1994) and FcεRI (Cook et al., 1997). In addition, the crystal structure of the human IgE-FcεRIα complex was recently solved by Garman and colleagues (Garman et al., 2000). The amino acid regions that are involved in receptor binding are localized in three loops and spread over most of the Cε3 domain (Pro-364, Arg-365, Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469). Binding is mediated primarily by electrostatic interaction.
Anti-IgE therapy is based on antibodies which bind the receptor-binding target domain Cε3 region of IgE, thereby preventing the binding of IgE to the FcεRI receptor and, therefore, preventing sensitization of mast cells and basophils. However, even if 99% of free IgE were neutralized by the anti-IgE antibody, the therapy still would fail because the few remaining IgE molecules would be sufficient to sensitize the respective cells. Therapeutic efficacy is provided through additional actions: FcεRI expression is regulated by the level of free IgE, in a way that reduced levels of free IgE lead to lowered densities of FcεRI on basophils and mast cells and lowered sensitivities. And, anti-IgE may lead to down-regulation of IgE production by eliminating or down-regulating IgE-expressing B cells, perhaps by cross-linking membrane-bound IgE and causing apoptosis, anergy or most likely also by complement-mediated and cell-mediated cytolysis (Wang et al., 2003). The latter mechanism was, however, not found in clinical trials performed with Omalizumab. For this monoclonal antibody, reduction of IgE production from B-cells (plasma cells) mediated by lowered IgE levels was only observed in animal and in-vitro experiments.
Most of the therapeutic monoclonal antibodies in development can only bind and neutralize free IgE or IgE associated with B-cells. In contrast, FcεRI-bound IgE is not accessible for these anti-IgE antibodies. Anti-IgE antibodies directed against regions of the IgE molecule outside of the receptor binding region (such as the variable, antigen-binding domain of IgE referred to as the IgE idiotype), can bind to an IgE molecule while it is bound to its receptor. This results in cross-linking of receptor-bound IgE, causing an anaphylactic shock in animals treated systemically with such antibodies. Importantly, except for defense mechanisms against parasite infections, IgE seems to play no role in normal physiology and IgE-deficient people are healthy with no apparent sign of pathology (Levy and Chen, 1970).
Omalizumab (XOLAIR®) is a humanized monoclonal anti-IgE antibody for passive immunization, and the first available/approved anti-IgE therapy on the market. A total of 7 phase III clinical trials were performed with this monoclonal anti-IgE antibody, which bind to the Cε3 region of IgE (for a review refer to (Bousquet et al., 2005) without crosslinking the FcεRI receptor. Omalizumab significantly reduced the rate of asthma exacerbations by 38% and the rate of total emergency visits by 47%. The efficacy of Omalizumab was unaffected by patient age, gender, baseline serum IgE or by 2- or 4-weekly dosing schedule, although benefit in absolute terms appeared to be greatest in patients with more severe asthma, defined by a lower value of percentage predicted forced expiratory volume in 1 s (FEV1) at baseline.
As outlined before, one disadvantage of passive immunization with a monoclonal antibody is the requirement of infusions every 2-4 weeks with relatively high antibody doses making such therapies expensive. Therefore, alternative approaches are needed for the treatment of allergic diseases such as atopic allergies or asthma.
According to the present invention this problem is solved by a structural protein of a parvovirus comprising an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/imimotope. Such structural proteins are preferably capable of forming virus-like particles. They harbor anti-idiotypic epi-/mimotopes of an anti-IgE antibody and/or IgE epi-/mimotopes on the surface of the capsid shell. Therefore, the anti-idiotypic epi-/mimotopes of an anti-IgE antibody, respectively the IgE epi-/mimotopes are accessible to the humoral immune system. Such structural protein can be used in patients to induce specifically an immune response against IgE, meaning antibodies that cross-react with IgE (anti-IgE antibodies), thereby preventing binding of IgE to its high affinity receptor FcRI.
For a lot of the publicly available therapeutic antibodies which can be used as target antibody for AAV selection, the epitopes are not known. To be able to compare the epitopes of the target antibodies and the antibodies induced in e.g. mice after vaccination, epitope mapping can be performed. For example, epitopes recognized by anti mouse or anti human IgE antibodies can be identified from arrays using overlapping peptide scans from the respective IgE spotted on nylon membranes. Preferred antibodies are those with a binding pattern similar to that of Omalizumab, that can be used for selection of mimitopes from the AAV capsid library. Epitopes recognized by antibodies induced in e.g. mice after vaccination can be identified from arrays spotted on glass slides. Cross-reactivity of anti human IgE antibodies or antibodies induced in mice after vaccination with the constant chain regions of other Ig's can be monitored in Westernblot experiments.
Especially preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain IgE epitopes or mimotopes, preferably previously known epitopes or mimotopes. As described by Rudolf, Stadler, Vogel and colleagues (Rudolf et al., 1998, Rudolf et al., 2000, Stadler et al., 1999, Vogel et al., 2000), one can develop so-called mimotope immunization vaccines based on peptide phage display libraries screened for particles recognizing BSW17, a mouse monoclonal anti-human IgE antibody. Peptide sequences best recognized by BSW17 are the mimotope sequences                EFCINHRGYWVCGD (‘Rudolf’ (Rudolf et al., 2000)), and (SEQ ID NO: 84)        INHRGYWV (‘C4M’, {Rudolf, 2000 #52}) (SEQ ID NO: 203)with G, W and V (underlined) being conserved among all sequences identified (the cysteine residues (in bold) mediate a circular form of the peptide via disulfide bridging) and the epitope        VNLTWSRASG (Kricek et al., 1999). (SEQ ID NO: 85)        
In the course of this invention previously novel epitopes that are especially suitable for vaccination purposes against allergic diseases like asthma have been identified:
(SEQ ID NO: 204)EKQRNGTLT (‘Bind2’) (SEQ ID NO: 205)EDGQVMDVDLS (‘Flex’) (SEQ ID NO: 206)TYQCRVTHPHLPRALMR (“3DEpi1”) (SEQ ID NO: 207)RHSTTQPRKTKGSG (“3DEpi2”) (SEQ ID NO: 208)DSNPRGVSAYLSR (“3DEpi3”) (SEQ ID NO: 209)TITCLVVDLAPSK (“3DEpi4”) (SEQ ID NO: 210)KTKGSGFFVF (“C4E”) (SEQ ID NO: 211)THPHLPRALMRS (“Wang-CS”) (SEQ ID NO: 212)GETYQCRVTHPHLPRALMRSTTK (“Wang”) (SEQ ID NO: 213)LPRALMRS (C21)
Accordingly, the present invention further relates to novel IgE B-cell epitopes Bind2, Flex, 3DEpi1, 3DEpi2, 3DEpi3, 3DEpi4, C4E, Wang-CS, Wang, and C21 and/or to a functionally active variant thereof. A functionally active variant of these epitopes means a B-cell epitope which generates in a rabbit vaccination experiment according to example 10.9 a B-cell response measurable as titer of specific antibodies binding to human IgE. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of allergic diseases, especially asthma.
Such functionally active variants can either be single peptides or mixtures of single peptides consisting of peptide sequences of up to 40 amino acids, preferably up to 25 amino acids, more preferably 15 amino acids, especially 9 amino acids of the given sequence, or a fusion of such functionally active variant to a carrier. Such carrier is meant to be any molecule except for the naturally occurring IgE protein or part thereof (larger than the functionally active variant), preferably a parvoviral particle, but also a different virus- or bacteriophage particle, a polymer (e.g. LPH) or a fusion protein, capable of generating a B cell response (as defined above) against such functionally active variant. Such fusion to a carrier can i.e. be obtained by chemically linking the variant to the carrier or by genetically making fusion proteins or insertion variants.
These and similar sequences or parts therefore including or excluding the cysteine residues and flanking sequences can be introduced into positions I-587 and others of AAV VP as described in FIG. 14. The corresponding AAV particles can be manufactured (initially as genome-containing infectious AAV), purified and characterized. Although AAV capsids have a different conformational structure than phages, the chance that a similar structure of the mimotope sequence EFCINHRGYWVCGD (SEQ ID NO: 84) is present on both, phages and AAV, is high due to the cysteine residues building up a loop structure of the peptide sequence. For linear epitopes such as VNLTWSRASG (SEQ ID NO: 85), interchangeability should also be possible. If these AAV particles bind BSW17 (the anti-IgE antibody used for phage display), they can be used as an anti-IgE vaccine that can be used with and without co-formulation in a suitable adjuvant.
Especially preferred embodiments of the invention are structural proteins of parvoviruses that contain IgE epi-/mimotopes that, once injected into an immunocompetent mammal, induce anti-IgE specific antibodies with therapeutic efficacy without cross-linking properties. Cross-linking properties means that in an immunocompetent mammal the generated anti-IgE antibodies are binding IgE molecules in a way that IgE/FcεRI binding is still possible. By such way, and if one antibody binds several IgE molecules at a time, the high-affinity FcεRI receptor is crosslinked on effector cells leading to its degranulation. This would induce a systemic anaphylactic shock. On the other hand, the structural proteins of parvoviruses should be able to directly crosslink the respective B-cell receptor (binding the IgE epi-/mimotopes or the anti-idiotype epi-/mimotope of an anti-IgE antibody) to activate the corresponding B-cells and to induce anti-IgE antibody production independent of a T-cell response.
Vaccines for the Treatment of Alzheimer's Disease
Especially preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain β-amyloid epitopes or mimotopes, preferably known epitopes or mimotopes, that can be used for the treatment of Alzheimer disease. In the context of the present invention a B-cell epitope of f-amyloid was inserted into a parvovirus capsid and displayed on the surface of the capsid. In a preferred embodiment the B-cell epitope is a human epitope. Preferably it is inserted into I-453 and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV-6. In an especially preferred embodiment the B-cell epitope has the sequence DAEFRHDSG (SEQ ID NO: 158).
In general, misfolded proteins leading to a protein aggregation and, therefore, causing conformational diseases, are good candidate targets for an active immunization approach with AAV vaccines. Ideally, B-cell epitopes represented by misfolded proteins or protein aggregates only are chosen for presentation on AAV particles (for an overview, please refer to Uversky et al., 2006, especially; table 1-1).
Vaccines for the Treatment of Atherosclerosis
Atherosclerosis is a disease affecting arterial blood vessels. It is a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low density (especially small particle) lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a “hardening” or “furring” of the arteries. It is caused by the formation of multiple plaques within the arteries. There is a strong inverse relationship between the plasma concentration of cholesterol in HDLs (HDL-C) and the development of coronary heart disease (CHD). Plasma concentration of HDL-C is a powerful predictor of CHD. Although 33% of patients with CHD have low plasma levels of HDL-C as their primary lipid abnormality, there is currently no effective therapy for increasing the plasma concentration of HDL-C. Diet and moderate exercise are ineffective, statins afford only a modest 5% to 7% increase in HDL-C, and niacin has side effects and compliance profiles that limit its use.
One therapeutic approach that has been suggested for increasing plasma HDL-C concentrations is the inhibition of cholesteryl ester transfer protein (CETP) activity. CETP is a 74-kDa plasma glycoprotein that facilitates transfer of neutral lipids and phospholipids between lipoproteins and contributes to the regulation of plasma concentration of HDL-C. CETP functions in the plasma to lower the concentration of HDL-C by moving cholesteryl esters from HDLs to VLDLs and LDLs (Rittershaus et al., 2000).
Accordingly it is one embodiment of the invention to provide structural proteins of parvoviruses, especially AAV, that contain CETP epitopes or mimotopes that can be used for the treatment of atherosclerosis. Suitable epitopes or mimotopes are the human CETP derived peptides hTP10, hTP11, hTP12, hTP13, hTP18 and hTP20, hRitsch-1, hRitsch-2, hRitsch-3, hCETP-intern and hCETP C-Term:
(SEQ ID NO: 214)PKTVSNLTESSSESVQS (hTP10) (SEQ ID NO: 215)SLMGDEFKAVLET (hTP11) (SEQ ID NO: 216)QHSVAYTFEED (hTP12) (SEQ ID NO: 217)INPEIITRDG (hTP13) (SEQ ID NO: 218)DISLTGDPVITASYL (hTP18) (SEQ ID NO: 219)DISLTGDPVITA (hTP20) (SEQ ID NO: 220)DQSIDFEIDSA (hRitsch-1) (SEQ ID NO: 221)KNVSEDLPLPTFSPTLLGDS (hRitsch-2) (SEQ ID NO: 222)KNVSEDLPLPT (hRitsch-3) (SEQ ID NO: 223)CDSGRVRTDAPD (hCETP-intern) (SEQ ID NO: 224)FPEHLLVDFLQSLS (hCETP C-Term)
The present invention further relates to novel CETP B-cell epitopes hTP10, hTP11, hTP12, hTP13, hTP18, hTP20, hRitsch-1, hRitsch-2, hRitsch-3, hCETP-intern and hCETP C-Term and/or to a functionally active variant thereof. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of atherosclerosis.
Vaccines for the Treatment of Tumor Diseases
Antibody therapies such as Herceptin, Avastin, Erbitux, Omnitarg, Rituxan, Campath, Zevalin, Mylotarg, Bexxar or Panitumumab play an increasing role in fighting various types of tumor diseases. These antibodies specifically bind epitopes of factors causing uncontrolled cellular growth, such as growth factor receptors or growth factors. Accordingly, it is a further embodiment of this invention to provide structural proteins of parvoviruses, especially AAV, that contain epitopes of such factors causing uncontrolled cellular growth.
HER2/neu (also known as ErbB-2, ERBB2) is a protein giving higher aggressiveness in breast cancers. It is a member of the ErbB protein family, more commonly known as the epidermal growth factor receptor family. HER2/neu has also been designated as CD340. HER2/neu is notable for its role in the pathogenesis of breast cancer and as a target of treatment. It is a cell membrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. Approximately 25-35 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression also occurs in other cancer such as ovarian cancer and stomach cancer. Clinically, HER2/neu is important as the target of the monoclonal antibody trastuzumab (marketed as Herceptin).
As for an active vaccination approach, the epitope sequence QMWAPQWGPD (SEQ ID NO: 225) presented in a circular way has been shown to induce polycloncal antibodies with therapeutic effectiveness. Therefore, an Her2/NEU-AAV vaccine can be generated by insertion of the peptide                QMWAPQWGPD (SEQ ID NO: 225)into AAV using suitable adaptor sequences (Riemer et al., 2007).Vaccines for the Treatment of Autoimmune Diseases and Chronic Inflammatory Diseases        
Autoimmune diseases as well as inflammatory diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body attacks its own cells.
Rheumatoid arthritis (RA) is an autoimmune disease which causes chronic inflammation of the joints, the tissue around the joints, as well as other organs in the body affecting 0.5-1.0% of the population in the industrialized world. It commonly leads to significant disability and consequently to a significant reduction of quality of life. If not treated appropriately, RA leads to a reduction of life expectancy (Smolen and Steiner, 2003).
Psoriasis is a chronic inflammatory disease of the skin characterized by overgrowth of epidermal cells, angiogenesis, infiltration of immune cells, and increased production of cytokines.
Similar activition of immune cells and increased production of cytokines is associated with autoimmune diseases and (chronic) inflammatory diseases as further listed below.
In order to limit or control such disease causing/related immune responses it has become an established therapeutic modality to neutralize cytokines involved in the pathogenesis of autoimmune and inflammatory diseases. Antibodies (infliximab, adalimumab) and a soluble receptor construct neutralizing the action of TNF-α (etanercept) have been established in the treatment of RA and other disease. Now there is evidence implicating several novel cytokines, including IL-32 and IL-17, in the pathogenesis of RA. In addition we assess the development of existing targets as they move towards clinical evaluation, particularly IL-1, IL-6, IL-15, IL-18 and the IL-12 superfamily (Asquith et al., 2007).
Vaccines for the Treatment of Infectious Diseases
Blocking of viral infection by induction of auto-antibodies against the cellular receptor of the virus is a suggested mechanism of a preventive or therapeutic vaccination against viruses, preferably for viruses where classical vaccination attemps have failed like HIV using CCR5 as the target receptor (Chackerian, 1999).
Accordingly, preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain epitopes or mimotopes of viral receptors, preferably of CCR5, preferably known epitopes or mimotopes that can be used as vaccines for the treatment of such viral infection and associated diseases, preferably HIV infection/AIDS. In a preferred embodiment the B-cell epitope is a human epitope.
Preferred B-cell epitopes are HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) or a functionally active variant thereof.
Accordingly, preferred embodiments of the invention are structural proteins of parvoviruses, especially AAV, that contain epitopes or mimotopes of cytokines, preferably of TNF-α, IL-6 and/or IL-17, preferably known epitopes or mimotopes, that can be used for the treatment of autoimmune diseases and/or chronic inflammatory diseases, preferably rheumatoid arthritis and/or Crohn's disease. In a preferred embodiment the B-cell epitope is a human epitope. Preferably it is inserted into I-453 and/or I-587, especially into I-453 and/or I-587 of AAV1, AAV2 or AAV-6. Preferred B-cell epitopes are the human epitopes:
(SEQ ID NO: 226)SSRTPSDKPVAHVVANPQAE (TNF-α V1) (SEQ ID NO: 227)SRTPSDKPVAHVVANP (TNF-α V2) (SEQ ID NO: 228)SSRTPSDKP (TNF-α V3) (SEQ ID NO: 229)NADGNVDYHMNSVP (IL-17 V1) (SEQ ID NO: 230)DGNVDYHMNSV (IL-17 V2) (SEQ ID NO: 231)RSFKEFLQSSLRALRQ (IL-6 V1) (SEQ ID NO: 232)FKEFLQSSLRA (IL-6 V2)
The present invention further relates to novel cytokine B-cell epitopes TNF-α V1, TNF-αV2, TNF-α V3, IL-17 V1, IL-17 V2, IL-6 V1 and IL-6 V2 and/or to a functionally active variant thereof. The invention further relates to medicaments in general comprising such epitopes or functionally active variants thereof, preferably vaccines comprising such epitopes or functionally active variants thereof for the treatment or prevention of autoimmune diseases and/or chronic inflammatory diseases, preferably rheumatoid arthritis, Crohn's disease or psoriasis.
According to this invention the structural proteins of parvoviruses are the viral capsid proteins that are referred to as VP-1, VP-2 and in many instances VP-3 for most of the known parvoviruses, especially the AAV. In principal the recombinant parvoviruses made from a mutant cap gene can be used directly for vaccination in animal models or even in humans. However, as such a vaccination is a gene therapy it is preferred to use inactivated (e.g. by gamma or UV-irradiation) genome-containing AAV particles, or virus-like particles of the respective parvovirus for vaccination purposes. Such virus-like particles are capsid-like structures that are composed of the structural proteins of the respective parvovirus, e.g. VP-1, VP-2 and/or VP-3, or parts thereof such as N- or C-terminal truncated structural proteins but do not contain a viral genome. VP-2 alone has been shown to assemble into virus-like particles and can be expressed in various expression systems such as bacteria e.g. E. coli, yeasts, e.g. Saccharomyces cerevisiae, hansenula polymorpha, Pichla pastoris, in insect cells, e.g. the baculovirus expression system (SF9, SF+ or High Five cells), or in mammalian cells (such as CHO, HeLa, 293, BHK, or PerC6).
Another preferred embodiment are structural proteins of parvoviruses that do not form regular virus-like particles but capsomers or other regular or amorphous aggregates that present the foreign epi-/mimotopes in a highly structured and/or dense manner.
The parvoviral mutated structural protein can further be fused to a second protein or peptide. Such second proteins can be tags, such as provided in Table 3. Tags can for example be used for purification purposes.
Preferably the parvoviral mutated structural protein is capable of forming a multimeric structure. Accordingly, another subject of the invention relates to a multimeric structure comprising parvovirus mutated structural proteins according to the invention. Such multimeric structure can be a capsomer, a virus-like particle or a virus. Capsomers are multimeric subunits of a viral capsid, typically consisting of 5-6 capsid proteins (pentamers and hexamers). Virus-like particles are empty viruses, meaning that they do not comprise genetic material such as a viral genome or relevant part thereof.
The multimeric structure may also be an aggregate of at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 structural proteins. Compared to capsomers or virus-like particles aggregates are amorphous structures with no symmetric order.
Preferably the B-cell epitope heterologous to the parvovirus is located on the surface of the multimeric structure.
A further embodiment of the present invention is a nucleic acid coding for a parvovirus mutated structural protein of the invention such as DNA, RNA, mRNA etc.
A further embodiment of the present invention is a virus that comprises a parvovirus mutated structural protein of the invention and or nucleic acid coding for a parvovirus mutated structural protein of the invention. Such virus may be active or inactive, for example it may have been inactivated through standard techniques such as attenuation or irradiation.
A further embodiment of the present invention is a cell comprising a nucleic acid coding for the parvovirus mutated structural protein. Such cell can be a bacterium, preferably E. coli, a yeast cell, preferably s. cerevisiae, hansenula polymorpha or pichia pastoris, an insect cell, preferably SF-9, SF+ or High5, or a mammalian cell, preferably HeLa, 293, VERO, PERC6, BHK or CHO.
The parvovirus mutated structural proteins of the invention can be prepared by the method comprising the steps of (a) expressing the nucleic acid coding for the parvovirus mutated structural protein by cultivating the cell as defined above under suitable conditions, and (b) isolating the expressed parvovirus mutated structural protein of step (a).
Another subject of the invention relates to a medicament, particularly a vaccine comprising at least one parvovirus mutated structural protein of the invention and/or a nucleic acid of the invention, preferably at least one multimeric structure of the invention. Preferably, the medicament is a vaccine.
In a preferred embodiment of the invention a vaccine is a mixture of more than one multimeric structures comprising parvovirus mutated structural proteins as further defined herein. Preferably two to three virus-like particles of a parvovirus displaying different B-cell epitopes as further defined herein are combined for the vaccination of a patient. Further, it is envisaged that a vaccine according to this invention is combined with some other type of vaccine for convenience of the patient.
The medicament of the present invention may further encompass pharmaceutically acceptable carriers and/or excipients. The pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the (poly)peptides herein disclosed. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e. g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
In a preferred embodiment the medicament further comprises an immunostimulatory substance such as an adjuvant. The adjuvant can be selected based on the method of administration and may include mineral oil-based adjuvants such as Freund's complete and incomplete adjuvant, Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containing muramyl dipeptide, or aluminum salt adjuvants. Preferably, the adjuvant is a mineral oil-based adjuvant, especially ISA206 (SEPPIC, Paris, France), most preferably ISA51 (SEPPIC, Paris, France). In another preferred embodiment the parvovirus mutated structural protein is co-formulated with at least one suitable adjuvant such as CpG, Imidazoquinolines, MPL, MDP, MALP; flagellin, LPS, LTA, or cholera toxin or derivative thereof, HSP60, HSP70, HSP90, saponins, QS21, ISCOMs, CFA, SAF, MF59, adamantanes, aluminum hydroxide, aluminum phosphate or a cytokine.
In a more preferred embodiment the immunostimulatory substance is selected from the group comprising polycationic polymers, especially polycationic peptides such as polyarginine, immunostimulatory deoxynucleotides (ODNs), peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, neuroactive compounds, especially human growth hormone, alumn, adjuvants or combinations thereof. Preferably, the combination is either a polycationic polymer and immunostimulatory deoxynucleotides or of a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides. In a still more preferred embodiment the polycationic polymer is a polycationic peptide.
In an even more preferred embodiment of the invention the immunostimulatory substance is at least one immunostimulatory nucleic acid. Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine dinucleotides (CpG) in a defined base context (e.g. as described in WO 96/02555). Alternatively, also nucleic acids based on inosine and cytidine as e.g. described in WO 01/93903, or deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 and WO 02/095027) may preferably be used as immunostimulatory nucleic acids in the present invention. Preferably, mixtures of different immunostimulatory nucleic acids are used in the present invention. Additionally, the aforementioned polycationic compounds may be combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones described in WO 01/93905, WO 02/32451, WO 01/54720, WO 01/93903, WO 02/13857 and WO 02/095027 and the AU application A 1924/2001.
In a further embodiment the medicament comprising the parvovirus mutated structural protein comprising at least one B-cell epitope heterologous to the parvovirus is (used for the manufacture of) a vaccine, preferably for preventing or treating an autoimmune disease (e.g. diabetes type 1), a tumor disease (examples are: melanoma: e.g. HMW MAA, glioblastome multiforme: e.g. CA125, anti-IL13R, colon cancer: e.g. CA125 or anti-EGF(R), breast cancer: e.g. Her2/NEU, ovarian cancer e.g. L1 adhesion molecule, B-cell lymphoma: e.g. CD20), an allergic disease (asthma, allergies such as allergic rhinitis, examples for targets are IgE, IL-4, IL-9, IL-13), a metabolic disease (e.g. high cholesterol, intervention into the cholesterol metabolism (target example: CETP), obesity, hypertension (target example: angiotensin II), an inflammatory disease (e.g. rheumatoid arthritis, Crohn's disease; target examples: IL-6, IL-17 and TNF-α), a neurological disease (e.g. Alzheimer's disease; target example: β-Amyloid) or to be used in ophthalmology (e.g. AMD; target example: VEGF).
Also encompassed by the present inventions are methods for vaccination and/or for treating or preventing the diseases specified herein by administering to a patient an effective amount of a parvovirus mutated structural protein of the invention and or nucleic acid coding for a parvovirus mutated structural protein of the invention.
Accordingly, a further aspect of the present invention relates to a medicament of of the invention for the treatment and/or prevention of    a) an allergic disease and/or asthma whereas the B cell epitope comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/mimotope, particularly a mimotope of sequence of EFCINHRGYWVCGD or INHRGYWV, with the first G, W and V being conserved and cysteine residues C mediating a circular form of the peptide via disulfide bridging, or particularly an epitope selected from the group consisting of EKQRNGTLT (SEQ ID NO: 204), EDGQVMDVDLS (SEQ ID NO: 205), TYQCRVTHPHLPRALMR (SEQ ID NO: 206), RHSTTQPRKTKGSG (SEQ ID NO: 207), DSNPRGVSAYLSR (SEQ ID NO: 208), TITCLVVDLAPSK (SEQ ID NO: 209), KTKGSGFFVF (SEQ ID NO: 210), THPHLPRALMRS (SEQ ID NO: 211), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212), LPRALMRS (SEQ ID NO: 213) and a functionally active variant thereof;    b) Alzheimer's disease whereas the B cell epitope comprises a β-amyloid epitope or mimotope, particularly comprising or having the sequence DAEFRHDSG (SEQ ID NO: 158) or a functionally active variant thereof;    c) atherosclerosis whereas the B cell epitope comprises a CETP epitope or mimotope, particularly an epitope selected from the group consisting of PKTVSNLTESSSESVQS (SEQ ID NO: 214), SLMGDEFKAVLET (SEQ ID NO: 215), QHSVAYTFEED (SEQ ID NO: 216), INPEIITRDG (SEQ ID NO: 217). DISLTGDPVITASYL (SEQ ID NO: 218), DISLTGDPVITA (SEQ ID NO: 219), DQSIDFEIDSA (SEQ ID NO: 220), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 221), KNVSEDLPLPT (SEQ ID NO: 222), CDSGRVRTDAPD (SEQ ID NO: 223), FPEHLLVDFLQSLS (SEQ ID NO: 224) and a functionally active variant thereof;    d) a tumor disease whereas the B cell epitope comprises a growth factor receptors or growth factors epitope or mimotope, particularly a HER2/neu epitope or mimotope, especially the epitope QMWAPQWGPD (SEQ ID NO: 225) or a functionally active variant thereof;    e) an autoimmune disease and/or chronic an inflammatory disease, preferably rheumatoid arthritis and/or Crohn's disease, whereas the κ cell epitope comprises an epitope or mimotope of a cytokine, preferably of TNF-α, IL-6 and/or IL-17, especially an epitope selected from the group consisting of SSRTPSDKPVAHVVANPQAE (SEQ ID NO: 226), SRTPSDKPVAHVVANP (SEQ ID NO: 227), SSRTPSDKP (SEQ ID NO: 228), NADGNVDYHMNSVP (SEQ ID NO: 229), DGNVDYHMNSV (SEQ ID NO: 230), RSFKEFLQSSLRALRQ (SEQ ID NO: 231), FKEFLQSSLRA (SEQ ID NO: 232) and a functionally active variant thereof; or    f) an infectious disease, preferably HIV infection, whereas the B cell epitope comprises an epitope or mimotope of a viral receptor, preferably of CCR5, especially an epitope selected from the group consisting of HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) and a functionally active variant thereof.
In a still further aspect of the present invention the medicament of the invention as specifid herein is used in a method of treating or preventing    a) an allergic disease and/or asthma whereas the B cell epitope comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE epi-/mimotope, particularly a mimotope of sequence of EFCINHRGYWVCGD or INHRGYWV, with the first G, W and V being conserved and cysteine residues C mediating a circular form of the peptide via disulfide bridging, or particularly an epitope selected from the group consisting of EKQRNGTLT (SEQ ID NO: 204), EDGQVMDVDLS (SEQ ID NO: 205), TYQCRVTHPHLPRALMR (SEQ ID NO: 206). RHSTTQPRKTKGSG (SEQ ID NO: 207), DSNPRGVSAYLSR (SEQ ID NO: 208), TITCLVVDLAPSK (SEQ ID NO: 209), KTKGSGFFVF (SEQ ID NO: 210), THPHLPRALMRS (SEQ ID NO: 211), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 212), LPRALMRS (SEQ ID NO: 213) and a functionally active variant thereof;    b) Alzheimer's disease whereas the B cell epitope comprises a β-amyloid epitope or mimotope, particularly comprising or having the sequence DAEFRHDSG (SEQ ID NO: 158) or a functionally active variant thereof;    c) atherosclerosis whereas the B cell epitope comprises a CETP epitope or mimotope, particularly an epitope selected from the group consisting of PKTVSNLTESSSESVQS (SEQ ID NO: 214), SLMGDEFKAVLET (SEQ ID NO: 215), QHSVAYTFEED (SEQ ID NO: 216), INPEIITRDG (SEQ ID NO: 217), DISLTGDPVITASYL (SEQ ID NO: 218), DISLTGDPVITA (SEQ ID NO: 219), DQSIDFEIDSA (SEQ ID NO: 220), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 221), KNVSEDLPLPT (SEQ ID NO: 222), CDSGRVRTDAPD (SEQ ID NO: 223), FPEHLLVDFLQSLS (SEQ ID NO: 224) and a functionally active variant thereof;    d) a tumor disease whereas the B cell epitope comprises a growth factor receptors or growth factors epitope or mimotope, particularly a HER2/neu epitope or mimotope, especially the epitope QMWAPQWGPD (SEQ ID NO: 225) or a functionally active variant thereof;    e) an autoimmune disease and/or chronic an inflammatory disease, preferably rheumatoid arthritis and/or Crohn's disease, whereas the B cell epitope comprises an epitope or mimotope of a cytokine, preferably of TNF-α, IL-6 and/or IL-17, especially an epitope selected from the group consisting of SSRTPSDKPVAHVVANPQAE (SEQ ID NO: 226), SRTPSDKPVAHVVANP (SEQ ID NO: 227), SSRTPSDKP (SEQ ID NO: 228), NADGNVDYHMNSVP (SEQ ID NO: 229), DGNVDYHMNSV (SEQ ID NO: 230), RSFKEFLQSSLRALRQ (SEQ ID NO: 231), FKEFLQSSLRA (SEQ ID NO: 232) and a functionally active variant thereof; or    f) an infectious disease, preferably HIV infection, whereas the B cell epitope comprises an epitope or mimotope of a viral receptor, preferably of CCR5, especially an epitope selected from the group consisting of HYAAAQWDFGNTMCQL (SEQ ID NO: 357), YAAQWDFGNTMCQ (SEQ ID NO: 358), RSQKEGLHYT (SEQ ID NO: 359) and a functionally active variant thereof,wherein an effective amount of the medicament is administered to a patient in need of the prevention or treatment.
The vaccine used for immunization may be administered to a subject in need thereof, preferably mammals, and still more preferably humans, in any conventional manner, Including oral, intranasal, intramuscular (i.m.), intra-lymph node, intradermal, intraperitoneal, subcutaneous (s.c.), and combinations thereof, but most preferably through intramuscular injection.
The volume of each dose for administration is preferably up to about 5 ml, still more preferably between 1 ml and 3 ml, and most preferably about 2 ml. The volume of the dose when intramuscular injection is the selected administration route is preferably up to about 5 ml, preferably up to 3 ml, preferably between 1 ml and 3 ml, more preferably between 0.5 ml and 2 ml, and most preferably about 1 ml. The amount of vaccine in each dose should be enough to confer effective immunity against and decrease the risk of developing clinical signs to a subject receiving a vaccination therewith.
Preferably, the unit dose of protein or nucleic acid should be up to about 5 μg protein/kg body weight, more preferably between about 0.2 to 3 μg, still more preferably between about 0.3 to 1.5 μg, more preferably between about 0.4 to 0.8 μg, and still more preferably about 0.6 μg. Alternative preferred unit doses could be up to about 6 μg protein or nucleic acid/kg body weight, more preferably between about 0.05 to 5 μg, still more preferably between about 0.1 to 4 μg.
The dose is preferably administered 1 to 4 times, especially 1 to 3 times, e.g. with an interval of 1 to 3 weeks. Preferred amounts of protein per dose are from approximately 1 μg to approximately 1 mg, more preferably from approximately 5 μg to approximately 500 μg, still more preferably from approximately 10 μg to approximately 250 μg and most preferably from approximately 25 μg to approximately 100 μg.
Nucleic acid delivery compositions and methods are known to those of skill in the art. The nucleic acid of the invention may be employed in the methods of this invention or in the compositions described herein as DNA sequences, either administered as naked DNA, associated with a pharmaceutically acceptable carrier or comprised in a vector. The nucleic may be administered therapeutically or as part of the vaccine composition e.g., by injection.
An “effective amount” of a nucleic acid composition may be calculated as that amount capable of exhibiting an in vivo effect, e.g. preventing or ameliorating a sign or symptoms. Such amounts may be determined by one of skill in the art. Preferably, such a composition is administered parenterally, preferably intramuscularly or subcutaneously. However, it may also be formulated to be administered by any other suitable route, including intra-nasally, orally or topically. The selection of the route of delivery and dosage of such therapeutic compositions is within the skill of the art.
Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
Examples for autoimmune disease that are especially suitable for this invention are listed in Table 5.
TABLE 5Autoimmune diseases and suitable antibody targets/antigensDiseaseantibody target/antigenMyasthenia gravisAcetylcholine receptorsGraves's diseaseThyroid-stimulating hormonereceptorThyroiditisThyroidInsulin-resistant diabetesInsulin receptorAsthmaBeta-2 adrenergic receptorsJuvenile insulin-dependent diabetesPancreatic islet cellsPernicious anemiaGastric parietal cellsAddison's diseaseAdrenal cellsIdiopathic hypoparathyroidismParathyroid cellsSpontaneous infertilitySpermPremature ovarian failureInterstitial cells, corpusluteum cellsPemphigusIntercellular substance of skinPrimary biliary cirrhosisMitochondriaAutoimmune hemolytic anemiaErythrocytesIdiopathic thrombocytopenic purpuraPlateletsIdiopathic neutropeniaNeutrophilsVitiligoMelanocytesOsteosclerosis and Meniere's diseaseType-II collagenChronic active hepatitisNuclei of hepatocytesGoodpasture's syndromeBasement membranesRheumatoid arthritisGamma globulin, virus-relatedantigens, IL-6, IL-17, TNF-αSjogren's syndromeNuclei and centromeresSystemic lupus erythematosusNuclei, DNA, RNA, erythrocytes,etc.SclerodermaNuclei and centromeresPolymyositisNuclei, RNA
Preferred autoimmune diseases are asthma, Juvenile insulin-dependent diabetes (diabetes type 1) and rheumatoid arthritis. Therefore, preferred antigens are the corresponding antigens of Beta-2 adrenergic receptors, Pancreatic islet cells, Gamma globulin E, virus-related antigens IL-6, IL-17, and TNF-α.
Examples for tumor diseases that are especially suitable for this invention are listed in Table 6.
TABLE 6Tumor diseases and suitable antibody targets/antigensDiseaseantibody target/antigenMelanomaHMW MAA (=high molecular weightmelanoma associated antigen), BAGE,GAGE, MAGE-3, Melan A, MART-1, NYESO, gp 100, tyrosinaseColon cancerCA125, EGFRGliobastome multiformeCA125, IL13R(GBM)Breast cancerHer2/NEUOvarian cancerL1 cell adhesion moleculevarious cancers (e.g. forVEGFcolon cancer, small lungcell carcinoma)B-cell lymphoma, e.g. Non-CD20Hodgkin Lymphoma
Examples for allergic diseases are asthma, especially atopic asthma, and all types of allergies. The preferred target antigens for vaccination against allergic diseases are IgE, IL9, and IL13, especially IgE.
An example for a metabolic disease is a disorder in the cholesterol metabolism (e.g. atherosclerosis), a preferred target antigen is CETP.
Examples for inflammatory diseases that are especially suitable for this invention are listed in Table 7.
TABLE 7(Chronic) Inflammatory diseasesDiseaseCOPD (chronic obstructive pulmonary disease)OA (osteoarthritis)Rheumatoid arthritisPolymyalgia rheumaticaGouty arthritis, Gout, PseudogoutAtherosclerosisCrohn's disease (inflammatory bowel disease)Shoulder tendinitis, BursitisColitisMultiple SclerosisSystemic Lupus ErythematosusPsoriasisJuvenile diabetesType I diabetes mellitus (insulin-resistant diabetes)HypothyroidismChronic fatigue syndromeKawasaki's diseaseCardiavascular diseasePericarditisLymph adenopathyRaynaud's phenomenonSarcoidosisSjogren's syndromeSpondyloarthropathiesVasculitidesSclerodermaGoodpasture's syndromeWegener's granulomatosistemporal = Giant cell arteritisCeliac diseaseAddison's diseaseAutoimmune hepatitisGrave's diseaseGraft-vs-host disease
Preferred target antigens are IL-6, IL-17, TNF-α and CD20.
Examples for diseases in ophthalmology are age-related macular degeneration (AMD) and diabetic retinopathy, a preferred target in these indications is VEGF.
Other preferred diseases are Alzheimer disease with the target antigen β-amyloid.
The parvovirus mutated structural protein comprising at least one B-cell epitope heterologous to the parvovirus can be especially useful for manufacture of a medicament for breaking immune tolerance.
In the context of the uses of the invention, the features of the parvovirus mutated structural protein are as defined above.
In a preferred embodiment the disease is not an infectious disease, meaning a disease caused by a virus, a bacterium, a fungus or a eukaryotic parasite.
In a further embodiment parvovirus mutated structural protein is not used to make a vector that is used in gene therapy.
In this document, the content of all cited documents is included by reference.
The following examples and figures are intended to explain the invention in detail without restricting it.